Association of kinesin with sensitivity to chemotherapeutic drugs

ABSTRACT

The invention provides diagnostic assays for assessing the sensitivity or resistance to DNA damaging agents and microtubule-directed chemotherapeutic drugs of malignant cells in a tumor or tissue. The assay provided involves determining gene expression levels of kinesin genes in the malignant cells, wherein under-expression of kinesin is found in cells resistant to DNA damaging agents and sensitive to microtubule-directed chemotherapeutic drugs, and over-expression of kinesin is found in cells sensitive to DNA damaging agents and resistant to microtubule-directed chemotherapeutic drugs.

This application is a divisional application of U.S. Ser. No.09/235,546, filed Jan. 22, 1999, now U.S. Pat. No. 6,043,340, issuedMar. 28, 2000, which is a divisional application of U.S. Ser. No.08/486,382, filed Jun. 7, 1995, now U.S. Pat. No. 5,866,327, issued Feb.2, 1999, which is a continuation of U.S. Ser. No. 8/177,571, filed Jan.5, 1994, now abandoned, which is a continuation-in-part of U.S. patentapplication Ser. No. 08/033,086, filed Mar. 3, 1993, which in turn is acontinuation-in-part of U.S. patent application Ser. No. 08/039,385,corresponding to International Patent Application Ser. No.PCT/US91/07492, filed on Oct. 11, 1991 and which entered the Nationalstage in the U.S. on Apr. 15, 1993, which is a continuation-in-part ofU.S. Ser. No. 07/599,730, filed Oct. 19, 1990, now U.S. Pat. No.5,217,889, issued Jun. 8, 1993.

This invention was made with government support under grants CA-56736-02by the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to genetic factors associated with sensitivity tochemotherapeutic drugs. More particularly, the invention relates tomethods for identifying such factors as well as to uses for suchfactors. The invention specifically provides genetic suppressor elementsderived from mammalian kinesin genes, and therapeutic and diagnosticuses related thereto.

2. Summary of the Related Art

A broad variety of chemotherapeutic agents are used in the treatment ofhuman cancer. For example the textbook CANCER: Principles & Practice ofOncology, 2d Edition, (De Vita et al., eds.), J.B. Lippincott Company,Philadelphia, Pa. (1985) discloses as major antineoplastic agents theplant alkaloids vincristine, vinblastine, and vindesine; the antibioticsactinomycin-D, doxorubicin, daunorubicin, mithramycin, mitomycin C andbleomycin; the antimetabolites methotrexate, 5-fluorouracil,5-fluorodeoxyuridine, 6-mercaptopurine, 6-thioguanine, cytosinearabinoside, 5-aza-cytidine and hydroxyurea; the alkylating agentscyclophosphamide, melphalan, busulfan, CCNU, MeCCNU, BCNU,streptozotocin, chlorambucil, bis-diaminedichloro-platinum,azetidinylbenzoquinone; and the miscellaneous agents dacarbazine, mAMSAand mitoxantrone.

These and other chemotherapeutic agents such as etoposide and amsacrinehave proven to be very useful in the treatment of cancer. Unfortunately,some tumor cells become resistant to specific chemotherapeutic agents,in some instances even to multiple chemotherapeutic agents. Such drugresistance or multiple drug resistance can theoretically arise fromeither the presence of genetic factors that confer resistance to thedrugs, or from the absence of genetic factors that confer sensitivity tothe drugs. The former type of factors have been identified, and includethe multiple drug resistance gene mdr-1 (see Chen et al., Cell 47:381-389). However, the latter type of factor remains largely unknown,perhaps in part because the absence of such factors would tend to be arecessive trait.

Identification of genes associated with sensitivity to chemotherapeuticagents is desirable, because the discovery of such genes can lead toboth diagnostic and therapeutic approaches for cancer cells and for drugresistant cancer cells, as well as to improvements in gene therapy andrational drug design. Recently, some developments have been made in thedifficult area of isolating recessive genetic elements, including thoseinvolved in cytotoxic drug sensitivity. Roninson et al., U.S. Pat. No.5,217,889 (issued Jun. 8, 1993) teach a generalized method for obtaininggenetic suppressor elements (GSEs), which are dominant negative factorsthat confer the recessive-type phenotype for the gene to which theparticular GSE corresponds. (See also Holzmayer et al., 1992, NucleicAcids Res. 20: 711-717). Gudkov et al., 1993, Proc. Natl. Acad. Sci. USA90: 3231-3235 teach isolation of GSEs from topoisomerase II cDNA thatinduce resistance to topoisomerase II-interactive drugs. Co-pending U.S.patent application Ser. No. 08/033,986, filed Mar. 3, 1993, disclosesthe discovery by the present inventors of a novel and unexpected resultof experiments performed to identify GSEs isolated from RNA of cellsresistant to the anticancer DNA damaging agent, etoposide. Thisreference discloses that a GSE encoding an antisense RNA homologous to aportion of a mouse kinesin heavy chain gene has the capacity to conferetoposide resistance to cells expressing the GSE. The experimentsdescribed in this reference also demonstrate that under-expression ofthe particular kinesin heavy chain gene disclosed therein was associatedwith naturally-occurring etoposide resistance in cultures ofdrug-selected human adenocarcinoma cells. These results wereparticularly unexpected because the role of kinesin genes in etoposideresistance was unknown in the art prior to the instant inventors'discoveries.

The kinesins comprise a family of motor proteins involved inintracellular movement of vesicles or macromolecules along microtubulesin eukaryotic cells (see Vale, 1987, Ann. Rev. Cell Biol. 3: 347-378;and Endow, 1991, Trends Biochem. Sci. 16: 221-225 for reviews). Amongthe family of kinesin genes are encoded kinesin light chains and kinesinheavy chains that assemble to form mature kinesin. A number of kinesingenes have been isolated in the prior art.

Gauger and Goldstein, 1993, J. Biol. Chem. 268: 13657-13666 disclosecloning and sequencing of a Drosophila kinesin light chain gene.

Navone et al., 1992, J. Cell. Biol. 117: 1263-1275 disclose cloning andsequencing of a human kinesin heavy chain gene.

Kato, 1991, J. Neurosci. 2: 704-711 disclose cloning and sequencing of amouse kinesin heavy chain gene.

Cyr et al., 1991, Proc. Natl. Acad. Sci. USA 88: 10114-10118 disclosecloning and sequencing of a rat kinesin light chain gene.

McDonald & Goldstein, 1990, Cell 61: 991-1000 disclose isolation of aDrosophila kinesin heavy chain gene.

Kosik et al., 1990, J. Biol. Chem. 265: 3278-3283 disclose isolation ofa squid kinesin heavy chain gene.

The present inventors have demonstrated that a heretofore unexpectedgene, a kinesin heavy chain gene, is involved in cellular sensitivity tothe anticancer drug etoposide, and that down-regulation of functionalexpression of this kinesin heavy chain gene is associated withresistance to this drug. Further experiments, disclosed herein, havesuggested that the role of kinesin genes in chemotherapeutic drugresistance may not be limited to this single member of the kinesin genefamily. These results further underscore the power of the GSE technologydeveloped by these inventors to elucidate unexpected mechanisms of drugresistance in cancer cells, thereby providing the opportunity and themeans for overcoming drug resistance in cancer patients. Reagents andmethods directed towards such goals are provided in this disclosure.

BRIEF SUMMARY OF THE INVENTION

The invention provides genetic suppressor elements (GSEs) that arerandom fragments derived from genes associated with sensitivity tochemotherapeutic drugs, and that confer resistance to chemotherapeuticdrugs and DNA damaging agents upon cells expressing such GSEs. Theinvention specifically provides GSEs derived from cDNA and genomic DNAencoding kinesin genes. Diagnostic assays useful in determiningappropriate candidate cancer patients bearing tumors likely to besuccessfully reduced or eliminated by administration of particularanticancer treatment modalities, including chemotherapeutic drugs andother DNA damaging agents, are provided by the invention, on the basisof levels of kinesin gene expression in the tumor cells borne by suchcancer patients. In vitro drug screening and rational drug designmethods are also within the scope of the instant disclosure.

The invention is based in part on the discoveries disclosed inco-pending U.S. patent application Ser. No. 08/033,086, filed Mar. 3,1993 and incorporated by reference, providing a method for identifyingand isolating GSEs that confer resistance to any chemotherapeutic drugfor which resistance is possible. Particularly provided herein aremethods for identifying GSEs derived from any kinesin gene, said GSEsbeing capable of conferring resistance to DNA damaging agents on cellsexpressing the GSEs. This method utilizes chemotherapeutic drugselection of cells that harbor clones from a random fragment expressionlibrary derived from kinesin-specific cDNA, and subsequent rescue oflibrary inserts from drug-resistant cells. In a second aspect, theinvention provides GSEs comprising oligonucleotides and/or peptidesderived from kinesin genes that function as GSEs in vivo and confer oncells expressing said GSEs resistance to DNA damaging agents, includingcertain chemotherapeutic drugs. In a third aspect, the inventionprovides a method for obtaining GSEs having optimized suppressoractivity for a kinesin gene associated with sensitivity to achemotherapeutic drug. This method utilizes chemotherapeutic drugselection of cells that harbor clones from a random fragment expressionlibrary derived from DNA of a kinesin gene associated with sensitivityto that chemotherapeutic drug, and subsequent rescue of the libraryinserts from drug resistant cells. Particularly and preferably providedare such optimized GSEs derived from a mouse or human kinesin gene. In afourth aspect, the invention provides synthetic peptides andoligonucleotides that confer upon cells resistance to DNA damagingagents, including certain chemotherapeutic drugs. These syntheticpeptides and oligonucleotides are designed based upon the sequence of adrug-resistance conferring GSE derived from a mouse or human kinesingene according to the invention.

In a fifth aspect, the invention provides a diagnostic assay for tumorcells that are resistant to one or more therapeutic DNA damaging agentsand, at the same time, sensitive to therapeutic anti-microtubularagents, due to the absence of expression or under-expression of akinesin gene. This diagnostic assay comprises quantitating the level ofexpression of any particular kinesin gene product in a particular tumorcell sample to be tested, and comparing the expression levels soobtained with a standardized set of cell lines expressing varyingamounts of kinesin gene mRNA and/or protein and having different degreesof resistance to chemotherapeutic drugs and DNA damaging agentsassociated with their levels of kinesin gene expression. In preferredembodiments, such a standardized set of cell lines is matched by tissuetype with the tissue type of the tumor cells to be evaluated.

In a sixth aspect, the invention provides methods for determining theappropriateness of candidates for particular cancer chemotherapeutictreatment modalities. In one preferred embodiment, the inventionprovides a means for determining whether a cancer patient is anappropriate candidate for treatment with DNA damaging chemotherapeuticdrugs or other DNA damaging agents such as radiation, the methoddetermining whether a kinesin gene, such as the kinesin heavy chain genedisclosed herein and in co-pending U.S. patent application Ser. No.08/033,086, is over-expressed or under-expressed in tumor cells borne bya cancer patient, relative to a standardized set of cell lines asdisclosed herein. Using this method, appropriate candidates fortreatment with DNA damaging agents, including certain chemotherapeuticdrugs, will be those patients whose tumor cells over-express the kinesingene. In another embodiment, the invention provides a means fordetermining whether a cancer patient is an appropriate candidate fortreatment with anti-microtubular chemotherapeutic drugs. Using thisaspect of the method, appropriate candidates for anti-microtubular agenttreatment will be those patients whose tumor cells under-express thekinesin gene compared with expression levels in a standardized set ofcell lines. In a particularly useful embodiment of this aspect of theinvention, potential candidate cancer patients for treatment withanti-microtubular anticancer agents will have failed or proven resistantto a course of cancer chemotherapy using DNA damaging agents.

In a seventh aspect, the invention provides a starting point for therational design of pharmaceutical products that are useful against tumorcells that are resistant to chemotherapeutic drugs. By examining thestructure, function, localization and pattern of expression of kinesingenes associated with resistance to DNA damaging agents and sensitivityto anti-microtubular chemotherapeutic drugs, strategies can be developedfor creating pharmaceutical products that will overcome drug resistancein tumor cells in which such kinesin genes are either over-expressed orunder-expressed.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2B shows a scheme for construction of a random fragmentexpression library (RFEL) from NIH 3T3 cDNA. FIG. 1 shows the overallconstruction scheme. FIG. 2B shows normalization of the cDNA fragments.In this panel, t represents total unfractionated cDNA, s and d representthe single-stranded and double-stranded fractions separated byhydroxyapatite, time points indicate the period of reannealing, andtubulin, c-myc, and c-fos indicate the probes used in Southernhybridization with the total, single-stranded and double-strandedfractions.

FIG. 2 shows the structure of the LNCX vector and the adaptor used incDNA cloning. The nucleotide sequences are shown for the ATG-sense(SEQ.ID.No.:1) and ATG-antisense (SEQ.ID.No.:2) strands of the adaptor.

FIG. 3 shows the overall scheme for selecting cell lines containingchemotherapeutic drug resistance-conferring GSEs and rescuing the GSEsfrom these cells.

FIG. 4A shows etoposide resistance conferred by preselected virus; FIG.4B shows PCR analysis of the selected and unselected population; FIG. 4Cdepicts the scheme for the GSE recloning experiment described in Example3.

FIGS. 5A and 5B shows resistance to various concentrations of etoposide,conferred upon the cells by the GSE anti-khcs under an IPTG-induciblepromoter (FIG. 5A), the scheme for this experiment (FIG. 5B).

FIG. 6 shows the nucleotide sequence of the GSE anti-khcs(SEQ.ID.No.:3.)

FIGS. 7A through 7C shows the nucleotide sequence of most of the codingregion of the mouse khcs cDNA (SEQ.ID.No.:4).

FIGS. 8A through 8D shows the dot matrix alignments of khcs proteinsequence deduced from the nucleotide sequence in FIGS. 7A through 7Cwith kinesin heavy chain sequences from human (FIG. 8A), mouse (FIG.8B), fruit fly (FIG. 8C), and squid (FIG. 8D).

FIGS. 9A and 9B illustrates the experimental protocol for drug-selectedproduction of kinesin-derived GSEs (FIG. 9A) and the structure of theadaptors used for the preparation of a random fragment KHCS cDNA library(FIG. 9B).

FIG. 10 shows etoposide resistance in HT1080 cells carrying insert-freevector virus or a random fragment library of human KHCS cDNA.

FIGS. 11A through 11G shows the effects of different drugs on 4-daygrowth of NIH 3T3 cells infected with insert-free vector virus or with avirus encoding anti-khcs. Cell growth in the absence of the drugdiffered less than 5% for the compared populations. Drug concentrationsare given in ng/mL. A representative series of parallel assays, carriedout in triplicate, is shown.

FIGS. 12A and 12B shows growth of cells carrying anti-khcs (solid lines)and control cells (broken lines) after treatment with colchicine orvinblastine. Cells were incubated with the drugs for 4 days, followed by2 or 4 days in drug-free media, as indicated. Cell growth, presented inarbitrary units, was evaluated by methylene blue staining.

FIGS. 13A through 13D shows a kinetic analysis of cell growth ofanti-khcs GSE-carrying cells (black lines) and control cells (greylines) incubated with different drugs. Cell growth was measured asdescribed for FIGS. 12A and 12B. Cells were plated and one day later(indicated by the first arrow) the indicated drugs were added atconcentrations as described. Four days later (indicated by the secondarrow), the drugs were removed from some of the plates. Solid linesindicate cell growth in the continuous presence of the drug, and brokenlines indicate cell growth after removal of the drug.

FIG. 14 demonstrates increased immortalization of primary mouse embryofibroblasts by infection with the LNCX vector containing the anti-khcsGSE, relative to cells infected with the LNCX vector alone or uninfected(control) cells.

FIG. 15 demonstrates increased immortalization of primary human skinfibroblasts by infection with the LNCX vector containing the anti-khcsGSE (left panel) at the 4th passage after infection, relative to humanskin fibroblasts in growth crisis (right panel).

FIG. 16 shows cDNA-PCR quantitative analysis of expression of the humankhcs gene in various unselected and etoposide-selected human HeLa cells.Lanes a shows results for clone CS(O), lands a′ for clone CX(200), lanesb for clone Σ/11(O), lanes b′ for clone Σ11 (1000), lanes c for clone6(O), lanes c′ for clone 6(1000), lanes d for clone Σ20(O) and lanes d′for clone Σ20 (1000). The numbers in parentheses for each clone nameindicate the concentration of etoposide (ng/ml) present in the growthmedia. Bands indicative of khcs expression are shown along with bandsfor β-2 macroglobulin expression as an internal control.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to means for suppressing specific gene functionsthat are associated with sensitivity and resistance to chemotherapeuticdrugs. The invention provides genetic suppressor elements (GSEs) derivedfrom kinesin genes that have such suppressive effect and thus conferresistance to DNA damaging agents including chemotherapeutic drugs. Theinvention further provides methods for identifying such GSEs, as well asmethods for their use.

For the purposes of this invention, the term “kinesin gene” will beunderstood to encompass any kinesin gene, particularly mammalian, andpreferably mouse or human, kinesin genes. Kinesins comprise a family ofrelated genes encoding a number of related motor proteins involved inintracellular movement of vesicles or macromolecules along microtubulesin eukaryotic cells (see Background of the Invention). The mature,functional kinesin molecule is comprised of products of a kinesin heavychain gene and a kinesin light chain gene. The instant inventionencompasses GSEs derived from both kinesin light chain and kinesin heavychain genes. The invention specifically is intended to contain withinits scope all kinesin genes and GSEs derived therefrom that are capableof causing resistance or sensitivity to DNA damaging agents.

The DNA damaging agents that fall within the scope of this invention areall DNA damaging agents, including but not limited to ionizing andultraviolet radiation, and certain chemotherapeutic drugs, includingamsacrine, etoposide, doxorubicin (Adriamycin), cisplatin, andcamptothecin.

In a first aspect, the invention provides a GSE derived from the cDNA ofa mouse kinesin heavy chain gene isolated from a normalized, randomfragment expression library made from total cellular mRNA from NIH 3T3cells and isolated on the basis of its ability to confer resistance tothe topoisomerase II drug, etoposide (described in Examples 1-4 hereinand co-pending U.S. patent application Ser. No. 08/033,086, filed Mar.3, 1993 and incorporated by reference). Prior to the discovery by thepresent inventors, there was no suspicion that kinesin was in any wayimplicated in etoposide sensitivity. These results demonstrated theability of the general method for identifying GSEs to provide much newand surprising information about the genetic basis for resistance tochemotherapeutic drugs.

In addition, the kinesin-derived GSE conferring resistance to etoposidecaused cellular effects suggesting that kinesin may be involved inprogrammed cell death. The method according to this aspect of theinvention therefore also provides valuable information about the geneticbasis for senescence and cell death. This may have importantimplications for studying genes involved in development, since GSEs usedto identify genes associated with chemotherapeutic drug resistance orsenescence can also be expressed as transgenes in embryos to determinethe role of such genes in development. The elucidation of the structureof the mouse kinesin heavy chain gene corresponding to this drugresistance-related GSE is described in Example 5 and functional analysesof the drug resistance capacity of this GSE is disclosed in Example 7.

In a second aspect, the invention provides a method for identifyingkinesin gene-derived GSEs that confer resistance to a DNA damagingagent. The GSEs identified by this method will be homologous to akinesin gene. For purposes of the invention, the term “homologous to” akinesin gene has two different meanings, depending on whether the GSEacts through an antisense mechanism or antigene mechanism (i.e., througha mechanism of interference at the protein level). In the former case, aGSE that is an antisense or antigene oligonucleotide or polynucleotideis homologous to a gene if it has a nucleotide sequence that hybridizesunder physiological conditions to the gene or its mRNA transcript byHoogsteen or Watson-Crick base-pairing. In the latter case, a GSE thatinterferes with a protein molecule is homologous to the gene encodingthat protein molecule if it has an amino acid sequence that is the sameas that encoded by a portion of the gene encoding the protein, or thatwould be the same, but for conservative amino acid substitutions. Ineither case, as a practical matter, whether the GSE is homologous to agene is determined by assessing whether the GSE is capable of inhibitingor reducing the function of the gene; in particular, any kinesin gene,preferably any mouse or human kinesin gene, as disclosed herein.

The method according to this aspect of the invention comprises the stepof screening a kinesin-specific cDNA or kinesin-specific genomic DNArandom fragment expression library phenotypically to identify clonesthat confer resistance to a DNA damaging agent such as certainchemotherapeutic drugs. Preferably, the library of random fragments ofkinesin-specific cDNA or kinesin-specific genomic DNA is cloned into aretroviral expression vector. In this preferred embodiment, retrovirusparticles containing the library are used to infect cells and theinfected cells are tested for their ability to survive in aconcentration of a DNA damaging agent that kills uninfected cells.Preferably, the inserts in the library will range from about 100basepairs (b.p.) to about 700 b.p. and more preferably, from about 200b.p. to about 500 b.p. Once a clonal population of cells that areresistant to the DNA damaging agent has been isolated, the library cloneencoding the GSE is rescued from the cells. At this stage, thenucleotide sequence of the insert of the expression library may bedetermined; in clones derived from a kinesin gene-specific cDNA randomfragment expression library, the nucleotide sequence is expected to behomologous to a portion of the kinesin gene cDNA nucleotide sequence.Alternatively, the rescued library clone may be further tested for itsability to confer resistance to DNA damaging agents and chemotherapeuticdrugs in additional transfection or infection and selection assays,prior to nucleotide sequence determination. Determination of thenucleotide sequence, of course, results in the identification of theGSE. This method is further illustrated in Example 6.

Thus, the invention provides a method for obtaining kinesin gene-derivedGSEs having optimized suppressor activity. By screening a randomfragment expression library made exclusively from kinesin gene-specificfragments, a much greater variety of GSEs derived specifically from thekinesin gene can be obtained, compared with a random fragment libraryprepared from total cDNA as in Example 1. Consequently, the likelihoodof obtaining optimized GSEs, i.e., those kinesin-derived GSEs conferringan optimal level of resistance to a chemotherapeutic drug, is maximizedusing the single gene random fragment library approach, as is shown ingreater detail in Example 6.

An additional feature of this aspect of the invention is the productionof a multiplicity of kinesin-specific GSEs by drug selection of cellsproducing infectious retroviral embodiments of the kinesin-derived GSEsof the invention. In this aspect, ecotropic cells infected with akinesin cDNA or kinesin genomic DNA-specific random fragment expressionlibrary are subjected to selection with a DNA damaging agent, preferablyand most practically a chemotherapeutic drug such as etoposide. Apopulation of resistant clones are thereby obtained, each containing adrug resistance-conferring, kinesin-derived GSE. Since these cells arecapable of producing infectious retroviral embodiments of the GSEs ofthe invention, a multiplicity of kinesin-derived GSEs, pre-selected forthe ability to confer drug resistance, can be easily and efficientlyproduced.

In a fourth aspect, the invention provides synthetic peptides andoligonucleotides that are capable of inhibiting the function of kinesingenes associated with sensitivity to chemotherapeutic drugs. Syntheticpeptides according to the invention have amino acid sequences thatcorrespond to amino acid sequences encoded by GSEs according to theinvention. Synthetic oligonucleotides according to the invention havenucleotide sequences corresponding to the nucleotide sequences of GSEsaccording to the invention. Once a GSE has been discovered andsequenced, and its orientation is determined, it is straightforward toprepare an oligonucleotide corresponding to the nucleotide sequence ofthe GSE (for antisense-oriented GSEs) or amino acid sequence encoded bythe GSE (for sense-oriented GSEs). In certain embodiments, suchsynthetic peptides or oligonucleotides may have the complete sequenceencoded by the GSE or may have only part of the sequence present in theGSE, respectively. In certain other embodiments, the peptide oroligonucleotide may have only a portion of the GSE-encoded or GSEsequence. In such latter embodiments, undue experimentation is avoidedby the observation that many independent GSE clones corresponding to aparticular gene will have the same 5′ or 3′ terminus, but generally notboth. This suggests that many GSE's have one critical endpoint, fromwhich a simple walking experiment will determine the minimum size ofpeptide or oligonucleotide necessary to inhibit gene function. Forpeptides, functional domains as small as 6-8 amino acids have beenidentified for immunoglobulin binding regions. Thus, peptides or peptidemimetics having these or larger dimensions can be prepared as GSEs. Forantisense oligonucleotides, inhibition of gene function can be mediatedby oligonucleotides having sufficient length to hybridize to theircorresponding mRNA under physiological conditions. Generally,oligonucleotides having about 12 or more bases will fit thisdescription. Preferably, such oligonucleotides will have from about 12to about 100 nucleotides. As used herein, the term oligonucleotidesincludes modified oligonucleotides having nuclease-resistantinternucleotide linkages, such as phosphorothioate, methylphosphonate,phosphorodithioate, phosphoramidate, phosphotriester, sulfone, siloxane,carbonate, carboxymethylester, acetamidate, carbamate, thioether,bridged phosphoramidate, bridge methylene phosphonate and bridgedphosphorothioate internucleotide linkages. The synthesis ofoligonucleotides containing these modified linkages is well known in theart. (See, e.g., Uhlmann and Peyman, 1990, Chemical Reviews 90: 543-584;Schneider and Banner, 1990, Tetrahedron Letters 31: 335). The termoligonucleotides also includes oligonucleotides having modified bases ormodified ribose or deoxyribose sugars.

In a fifth aspect, the invention provides dominant selectable markersthat are useful in gene co-transfer studies. Since GSEs according to theinvention confer resistance to chemotherapeutic drugs, the presence of avector that expresses the GSE can readily be selected by growth of avector-transfected cell in a concentration of the appropriate cytotoxicdrug that would be cytotoxic in the absence of the GSE. GSEs accordingto the invention are particularly well suited as dominant selectablemarkers because their small size allows them to be easily incorporatedalong with a gene to be co-transferred even into viral vectors havinglimited packaging capacity.

In a sixth aspect, the invention provides a diagnostic assay for tumorcells that are resistant to one or more DNA damaging agents, includingcertain chemotherapeutic drugs, due to the absence of expression or theunder-expression of a kinesin gene. In particular, the class of DNAdamaging agents resistance to which involves under-expression of akinesin gene includes but is not limited to cisplatin, etoposide andcamptothecin. To determine whether absence of expression orunder-expression of a kinesin gene is a naturally occurring, and thusmedically significant basis for chemotherapeutic drug resistance, humantumor cells can be treated with cytotoxic quantities of an appropriatechemotherapeutic drug to select for spontaneous drug resistant mutants.These mutants can then be assessed for their level of expression of theparticular gene of interest. Absence of expression or significantlyreduced expression indicates a natural mechanism of chemotherapeuticdrug resistance. The description of such an experiment, disclosing thatunder-expression of the human kinesin heavy chain gene disclosed hereinis associated with naturally-occurring resistance to thechemotherapeutic drug etoposide in cultures of etoposide-resistant humanadenocarcinoma (HeLa) cells, is disclosed in Example 11 herein and inco-pending U.S. patent application Ser. No. 08/033,086, filed Mar. 3,1993 and incorporated by reference. In a preferred embodiment of thisassay, a standardized set of tissue-specific cell lines, wherein thelevels of drug resistance and kinesin gene expression have beenquantitated and correlated with each other, are provided for tumors fromeach tissue type to be assayed.

Alternatively, and preferably, collections of naturally occurringtreatment-responding and non-responding tumor tissue samples can beexamined for expression levels of kinesin genes, and correlationsestablished between treatment outcome, and presumably the drug-resistantmechanisms thereof, and kinesin gene expression.

Accordingly, such reduced or absent expression can be the basis for adiagnostic assay for tumor cell resistance to a DNA damaging agent orchemotherapeutic drug or drugs of interest. A first embodiment of adiagnostic assay according to this aspect of the invention utilizes anoligonucleotide or oligonucleotides that is/are homologous to thesequence of a kinesin gene. In this embodiment, RNA is extracted from atumor sample, and RNA specific for a particular kinesin gene isquantitated by standard filter hybridization procedures, an RNaseprotection assay, or by quantitative cDNA-PCR (see Noonan et al., 1990,Proc. Natl. Acad. Sci. USA 87: 7160-7164). In a second embodiment of adiagnostic assay according to this aspect of the invention, antibodiesare raised against a synthetic peptide having an amino acid sequencethat is identical to a portion of the kinesin heavy chain or kinesinlight chain protein. Antibodies specific for the human kinesin heavychain have in fact been disclosed (see Navone et al., supra). Theseantibodies are then used in a conventional quantitative immunoassay(e.g., RIA or immunohistochemical assays) to determine the amount of thegene product of interest present in a sample of proteins extracted fromthe tumor cells to be tested, or on the surface or at locations withinthe tumor cells to be tested.

A particular utility for such diagnostic assays of this invention istheir clinical use in making treatment decisions for the alleviation ofmalignant disease in humans. For example, a determination that thekinesin heavy chain gene of this invention is under-expressed in a tumorcompared with the levels of expression found in normal cells comprisingthat tissue would suggest that a patient bearing such a tumor might be apoor candidate for therapeutic intervention using DNA damaging agents,since it would be expected that such kinesin under-expressing cells ofthe tumor would be resistant to such agents. Similarly, tumor cellswhich fortuitously over-express the kinesin heavy chain gene of theinvention would be expected to be sensitive to such agents and thus tobe susceptible to tumor cell killing by these agents. On the other hand,the instant disclosure provides experimental evidence that kinesin heavychain gene under-expressors are sensitive to the cytocidal action ofanti-microtubular agents, including for example colchicine, colcemide,vinblastine, vincristine and vindesine. These results suggest thatpatients bearing tumors whose cells under-express the kinesin heavychain gene of the present invention may be responsive to treatment withanti-microtubular agents. The present invention thus enables intelligentand informed therapeutic intervention based on properties of anindividual cancer patient's tumor resistance or sensitivity to DNAdamaging agents and other chemotherapeutic treatment modalities, wheretreatment choices can be made prior to initiation of treatment based onmechanisms of resistance specific for DNA damaging agents and mediatedby kinesin heavy chain gene over- or under-expression. Particularlyuseful in this aspect of the invention are kinesin-specific antibodies,such as the anti-kinesin heavy chain antibodies described in Navone, etal., supra, for detection of kinesin expression levels in tumor samples.

In a seventh aspect, the invention provides a starting point for therational design of pharmaceutical products that can counteractresistance by tumor cells to chemotherapeutic drugs.

Understanding the biochemical function of the kinesin genes that areinvolved in drug sensitivity is likely to suggest pharmaceutical meansto stimulate or mimic the function of such genes and thus augment thecytotoxic response to anticancer drugs. One may also be able toup-modulate gene expression at the level of transcription. This can bedone by cloning the promoter region of each of the corresponding kinesingenes and analyzing the promoter sequences for the presence of ciselements known to provide the response to specific biologicalstimulators. Due to the structure of the kinesins in eukaryotic cells,i.e., comprised of both kinesin heavy chain and kinesin light chainproteins, coordinate up-regulation of the expression of both of theappropriate kinesin light chain and heavy chain proteins would berequired for efficacious therapeutic intervention based on modulatingexpression of kinesin genes.

Alternatively, kinesin expression in a cancer cell can be increased byco-introduction of recombinant expression constructs encodingfunctional, full-length copies of a kinesin heavy chain and a kinesinlight chain, whereby coordinate co-expression of such exogenous kinesinswould result in increased expression of functional kinesin molecules inthe cancer cells.

The protein structure deduced from the cDNA sequence can also be usedfor computer-assisted drug design, to develop new drugs that affect thisprotein in the same manner as the known anticancer drugs. The purifiedprotein, produced in a convenient expression system, can also be used asthe critical component of in vitro biochemical screen systems for newcompounds with anticancer activity. Accordingly, mammalian cells thatexpress chemotherapeutic drug resistance-conferring GSEs according tothe invention are useful for screening compounds for the ability toovercome drug resistance. As with pharmaceutical intervention methods,both kinesin light chains and heavy chains should be present in such invitro screening systems in amounts capable of reconstituting maturekinesin molecules in vitro.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

EXAMPLE 1 Generation of a Normalized Random Fragment cDNA Library in aRetroviral Vector

A normalized cDNA population was prepared using a modification of theprotocol of Patanjali et al. (1991, Proc. Natl. Acad. Sci. USA 88:1943-1947), illustrated in FIG. 1. Poly(A)⁺ RNA was extracted from NIH3T3 cells. To obtain mRNAs for different genes expressed at variousstages of the cell growth, one half of the RNA was isolated from arapidly growing culture and the other half from quiescent cells that hadreached complete monolayer confluence. To avoid overrepresentation ofthe 5′-end sequences in a randomly primed cDNA population, RNA wasfragmented by boiling to an average size range of 600-1,000 nucleotides.These RNA fragments were then used for preparing randomly primeddouble-stranded cDNA. This randomly primed cDNA was then ligated to asynthetic adaptor providing ATG codons in all three possible readingframes and in a proper context for translation initiation. The structureof the adaptor (see FIG. 2) determined its ligation to the blunt-endedfragments of the cDNA in such a way that each fragment started frominitiation codons independently from its orientation. The adaptor wasnot supplied with termination codons in the opposite strand since thecloning vector pLNCX, contained such codons immediately downstream ofthe cloning site. (This vector has been described by Miller and Rosman,1989, Biotechniques 7: 980-986.) The ligated mixture was amplified byPCR, using the “sense” strand of the adaptor as a PCR primer, (incontrast to the method of Patanjali et al., which utilized cloning theinitial cDNA preparation into a phage vector and then usingvector-derived sequences as PCR primers to amplify the cDNA population.)The PCRs were carried out in 12 separate reactions that weresubsequently combined, to minimize random over- or under-amplificationof specific sequences and to increase the yield of the product. ThePCR-amplified mixtures was size-fractionated by gel electrophoresis, and200-500 bp fragments were selected for subsequent manipulations, (incontrast to Patanjali's fragment size range of from 400 to 1,600 bp.)

For normalization, the cDNA preparation was denatured and reannealed,using different time points for reannealing, as described by Patanjaliet al., supra, and shown in FIG. 1. The single-stranded anddouble-stranded DNAs from each reannealed mixture were separated byhydroxyapatite chromatography. The single-stranded DNA fractions fromeach time point of reannealing were PCR-amplified using theadaptor-derived primer and analyzed by Southern hybridization for therelative abundance of different mRNA sequences. The fraction thatcontained similar proportions of tubulin, c-myc and c-fos cDNA sequences(see FIG. 2B), corresponding to high-, medium- and low-expressed genes,respectively, was used for the library preparation.

The normalized cDNA preparation was cloned into a ClaI site of theMoMLV-based retroviral vector pLNCX, which carries the neo (G418resistance) gene, transcribed from the promoter contained in theretroviral long terminal repeat (LTR), and which expresses the insertedsequence from a strong promoter of the cytomegalovirus (CMV) (see FIG.2). The ligation mixture, divided into five portions, was used for fivesubsequent large-scale transformations of E. coli. The transformedbacteria were plated on the total of 500 agar plates (150 mm indiameter) and the plasmid population (18 mg total) was isolated from thecolonies washed off the agar. A total of approximately 5×10⁷ clones wereobtained, more than 60% of which carried the inserts of normalized cDNA,as estimated by PCR amplification of inserts from 50 randomly pickedcolonies. These results demonstrate the feasibility of generating anormalized cDNA library of as many as 3×10⁷ recombinant clones in aretroviral plasmid expression vector.

EXAMPLE 2 Transduction of a Retroviral Random Fragment Library intoVirus-Packaging Cell Lines and NIH 3T3 Cells

The plasmid library prepared according to Example 1 was converted into amixture of retroviral particles by transfection into virus-packagingcells (derivatives of NIH 3T3) that express retroviral virion proteins.(Examples of such cell lines have been described by Markowitz et al.,1988, Virology 167: 400-406.) Ecotropic and amphotropic virus-packagingcell lines, GP+E86 and GP+envAm12, respectively, were mixed at a 1:1ratio and 10⁷ cells of this mixture were transfected with the plasmidlibrary under standard calcium phosphate coprecipitation conditions.This transfection resulted in the packaging and secretion of ecotropicand amphotropic virus particles, which rapidly spread through thepackaging cell population, since ecotropic viruses are capable ofinfecting amphotropic packaging cells and vice versa. The yield of thevirus, as measured by the number of G418-resistant colonies obtainedafter the infection of NIH 3T3 cells, reached 10⁵ infectious units per 1mL of media during the stage of transient transfection (1-3 days), thendecreased (4-8 days) and then rapidly increased due to the expression ofproviral genomes that became stably integrated in most of the packagingcells. The yield of the virus 9-12 days after transfection reached >10⁶per 1 mL of media supernatant. At this stage, the library showed fairlyeven representation of different fragments, but at later stagesindividual virus-producing clones began to predominate in thepopulation, leading to uneven representation of cDNA-derived inserts.The uniformity of sequence representation in the retroviral populationwas monitored by rapid extraction of DNA from cells infected with thevirus-containing supernatant, followed by PCR amplification of inserts.The inserts were analyzed first by the production of a continuous smearin ethidium bromide-stained agarose gel and then by Southernhybridization with different probes, including topoisomerase II, c-mycand tubulin. As long as each gene was represented by a smear of multiplefragments, the representativity of the library was considered to besatisfactory.

In other experiments, for transducing the random-fragment normalizedcDNA library into NIH 3T3 cells, without loss of representativity, NIH3T3 cells were infected either with a virus produced at the transientstage of transfection (days 1-3), or with the high-titer virus collected10-12 days after transfection. In the latter case, 100 ml of viralsuspension contained more than 10⁸ infectious units. In the case of the“transient” virus, NIH 3T3 cells were infected with at least 10⁷recombinant retroviruses by using 500 ml of media from virus-producingcells (five rounds of infection, 100 ml of media in each). These resultsdemonstrate the feasibility of converting a large and complex randomfragment library into retroviral form and delivering it to anon-packaging cell line without loss of complexity.

EXAMPLE 3 Isolation of GSEs Conferring Resistance to theChemotherapeutic Drug Etoposide

The overall scheme for the selection of GSEs conferring etoposideresistance is illustrated in FIG. 3. This selection was carried outdirectly on virus-producing packaging cells, in the expectation thatcells whose resistant phenotype is caused by the GSE expression willproduce virus particles carrying such a GSE. The mixture of amphotropicand ecotropic packaging cells was transfected with the cDNA library inthe LNCX vector, prepared according to Example 1, and the virus wasallowed to spread through the population for 9 days. Analysis of a smallpart of the population for G418 resistance showed that practically 100%of the cells carried the neo-containing provirus. The cells were thenexposed to 350 ng/mL etoposide for 15 days and then allowed to growwithout drug for two more weeks. No difference was observed between thenumbers of colonies obtained in the experiment and in the control(uninfected cells or cells infected with the insert-free LNCX virus)after etoposide selection. The virus present in the media supernatant ofthe surviving cells was then used to infect NIH 3T3 cells followed byetoposide selection using essentially the same protocol. NIH 3T3 cellsinfected with the library-derived virus produced by packaging cells thatwere selected with etoposide showed a major increase in the number ofetoposide-resistant cells relative to the control cells infected withthe insert-free LNCX virus, indicating the presence of biologicallyactive GSEs in the preselected virus population (see FIG. 4A).

The proviral inserts contained in the etoposide-selected NIH 3T3 cellswere analyzed by PCR. This analysis (see FIG. 4B) showed an enrichmentfor specific fragments, relative to the unselected population of theinfected cells. Individual PCR-amplified fragments were recloned intothe LNCX vector in the same position and orientation as in the originalplasmid, as illustrated in FIG. 4C. A total of 42 proviral inserts,enriched after etoposide selection, were thus recloned, and testedeither in batches or individually for the ability to confer increasedetoposide resistance after retroviral transduction into NIH 3T3 cells.Three non-identical clones were found to induce etoposide resistance,indicating that they contained biologically active GSEs. These GSEs weenamed anti-khcs, VPA and VP9-11. Etoposide resistance induced by theclone named anti-khcs is illustrated in FIG. 5A.

The ability of one of the anti-khcs GSE to induce etoposide resistancewas further documented by using the isopropyl β-D-thiogalactopyranoside(IPTG)-inducible promoter/activator system, as described by Baim et al.(1991, Proc. Natl. Acad. Sci. USA 88: 5072-5076). The components of thissystem include an enhancer-dependent promoter, combined in cis withmultiple repeats of the bacterial lac operator, and a gene expressingLAP265, an artificial regulatory protein derived from the lac repressorand a mammalian transcriptional activator. The anti-khcs GSE was clonedinto the plasmid pX6.CLN, which contains the inducible promoter used byBaim et al., supra (a gift of Dr. T. Shenk) which expresses the insertsfrom an enhancerless SV40 early gene promoter supplemented with 21repeats of the lac operator sequence. The resulting plasmid, whichcontains no selectable markers, was co-transfected into NIH 3T3 cellstogether with the LNCX plasmid carrying the neo gene. The masspopulation of G418- selected transfectants, along with control cellstransfected with the insert-free vector, was exposed to increasingconcentrations of etoposide, in the presence or in the absence of 5 mMIPTG. Even though the co-transfection protocol usually leads to theintegration of the GSE in only a fraction of the G418-resistant cells,transfection with anti-khcs resulted in a clearly increased etoposideresistance, which was dependent on IPTG (see FIG. 5B).

EXAMPLE 4 Sequence Analysis of GSEs Conferring Resistance to theChemotherapeutic Drug Etoposide

The GSE anti-khcs, cloned as described in Example 3, was sequenced bythe standard dideoxy sequencing procedure, and the deduced sequence isshown in FIG. 6. The nucleotide sequence of the “sense” and “antisense”strands, as well as amino acid sequence of the predicted peptidesencoded by each of these strands, were analyzed for homology to thenucleic acid and protein sequences present in the National Center forBiotechnology Information data base, using the BLAST network program forhomology search. The sequence corresponding to the “antisense” strand ofthe anti-khcs GSE, showed strong homology with several genes encodingthe heavy chain of kinesins, a family of microtubule motor proteinsinvolved in intracellular movement of organelles or macromolecules alongthe microtubules of eukaryotic cells. The highest homology was foundwith the human kinesin heavy chain (KHC) gene, as described by Navone etal. (1992, J. Cell Biol. 117: 1263-1275). Anti-khcs therefore encodesantisense RNA for a mouse khc gene, which we have termed khcs for khcassociated with sensitivity (to drugs) or senescence. We refer to thekinesin molecule, formed by the associate of the KHCS protein withkinesin light chains, as kinesin-S, to distinguish it from the otherkinesins present in mammalian cells. These results demonstrate thatchemotherapeutic drug selection for GSEs can lead to the discovery ofnovel genetic elements, and can also reveal roles of genes in drugsensitivity that had never before been suspected.

EXAMPLE 5 Cloning and Analysis of the Gene from Which Anti-khcs GSE Genewas Derived

The anti-khcs GSE isolated in Example 3 was used as a probe to screen400,000 clones from each of two cDNA libraries in the lambda gt10vector. These libraries were prepared by conventional procedures fromthe RNA of mouse BALB/c 3T3 cells, either unsynchronized or at G₀→G₁transition, as described by Lau and Nathans (1985, EMBO J. 4: 3145-3151and 1987, Proc. Natl. Acad. Sci. USA 84: 1182-1186, a gift of Dr. L.Lau). Screening of the first library yielded no hybridizing clones, buttwo different clones from the second library were found to containanti-khcs sequences. These clones were purified and sequenced. Sequenceanalysis showed that we have isolated the bulk of the mouse khcs cDNA,corresponding to 796 codons (the full-length human KHC cDNA encodes 963amino acids). This sequence is shown in FIGS. 7A through 7C, anadditional 252 nucleotides encoding 84 amino acids from the aminoterminus have been determined from 5′-specific cDNA isolated using the“anchored PCR” technique, as described by Ohara et al. (1989, Proc.Natl. Acad. Sci. USA 86: 5763-5677). Additional missing 3′terminalsequences are currently being isolated using this technique.

The dot-matrix alignment of the sequenced portion of the khcs proteinwith previously cloned KHC proteins from the human (see Navone et al.,1992, J. Cell, Biol. 117: 1263-1275), mouse (see Kato, 1991, J.Neurosci. 2: 704-711), Drosphila (McDonald & Goldstein, 1990, Cell 61:991-1000), and squid (see Kosik et al., 1990, J. Biol. Chem. 265:3278-3283) is shown in FIGS. 8A through 8D. The portion corresponding tothe anti-khcs GSE, is shown in brackets. The khcs gene is most highlyhomologous to the human gene (97% amino acid identity), suggesting thatthe human KHC (KHCS) gene is functionally equivalent to the mouse khcs.The alignment also shows that the anti-khcs GSE corresponds to theregion which is the most highly diverged between different kinesins(shown in the Figure by brackets around these sequences).

EXAMPLE 6 Generation of a Random Fragment KHCS cDNA Retroviral Libraryand Isolation of KHCS-derived GSEs

As described in Example 5 above, the murine khcs gene is highlyhomologous to the human KHC (or KHCS) gene described by Navone et al.,(1992, J. Cell Biol, 117: 1263-1275). The functional equivalence ofthese genes was also suggested by the observation that the levels ofKHCS mRNA are decreased in human cells selected for etoposide resistance(see Example 8). To determine conclusively that the human KHCS generepresents the functional equivalent of the mouse khcs, it wasdetermined whether any random fragment of human KHCS cDNA could functionas an etoposide-resistance GSE.

A library of random DNAaseI-generated fragments of a full-length humanKHCS cDNA (2.9 kb in length; provided by Dr. R. Vale, University ofCalifornia at San Francisco) was generated essentially as describedabove for topoisomerase II cDNA (see Example 1 in co-pending U.S. patentapplication Ser. No. 08/033,086, incorporated by reference), using theprotocol illustrated in FIG. 9A, with the following modifications.Specifically, two synthetic adaptors, instead of one, were used forligation with DNAase I-generated cDNA fragments. One adaptor, containingthree ATG codons, carried a HindIII cloning site (FIG. 9B). The otheradaptor had translation stop codons in all three reading frames andcarried a ClaI cloning site (FIG. 9B). After ligation with the equimolarmixture of both adaptors, cDNA fragments were amplified by PCR usingsense and antisense strands of the first and second adaptor,respectively. PCR products were digested with ClaI and HindIII andcloned into the corresponding sites of the pLNCX plasmid. Thismodification of the cloning strategy resulted in avoiding the formationof inverted repeats at the ends of the cDNA inserts after cloning intothe retroviral vector.

A plasmid library of 20,000 independent insert-carrying clones wasobtained and transfected into ecotropic packaging cells using thecalcium phosphate precipitation technique. Virus released by transientlytransfected cells was used to infect HT 1080/pJET-2TGH cells, clone 2, aderivative of human HT1080 fibrosarcoma cell line transfected with aplasmid expressing the murine ecotropic receptor (Albritton et al.,1989, Cell 57: 659-666) and susceptible to infection with ecotropicretroviruses (provided by Dr. G. R. Stark, Cleveland Clinic Foundation).After infection and G418 selection, these cells (further referred to asHT1080/ER) were plated at 10⁵ cells per 100 mm plate and cultivated for12 days in different concentrations of etoposide (200-500 ng/mL). Afterremoval of the drug, cells were allowed to grow in media without drugfor 7 more days. At this point, some of the plates were fixed andstained with crystal violet, to determine the number of survivingcolonies. (FIG. 10). As illustrated in FIG. 10 at drug concentrations250 ng/mL etoposide, there were only several colonies in control plates,compared with about a hundred times more colonies in the platescontaining GSE-containing cells.

In a parallel experiment, virus-producing mixtures of packaging cellswere subjected to similar etoposide selection. At all drugconcentrations tested, there were many more colonies surviving etoposidetreatment in the GSE-carrying cells than in the control cell population.

These results indicated that the retroviral library of random fragmentsof KHCS cDNA contained numerous GSEs inducing drug resistance in humancells, confirming that human KHCS is associated with drug resistance.Some of these GSEs are likely to be more potent as selectable markers ofdrug resistance that the original single GSE from the murine khcs gene.The virus isolated from such etoposide-resistant cells represents acollection of a multiplicity of kinesin-derived, drugresistance-conferring GSEs, which multiplicity is itself an aspect ofthe present invention and is useful in conferring resistance to DNAdamaging agents, including chemotherapeutic drugs, as disclosed herein.

EXAMPLE 7 Generation of Cell Populations Carrying Multiple Copies ofanti-khcs GSEs

A 1:1 mixture of ecotropic and amphotropic packaging cells wastransfected with retroviral vector pLNCX carrying the anti-khcs GSEusing a standard calcium phosphate procedure. Two weeks later, the virustiter, as measured by the formation of G418- resistance NIH 3T3colonies, reached >10⁶ infectious units per mL as a result of“ping-pong” infection (see Bodine et al., 1990, Proc. Natl. Acad. Sci.USA 87: 3738-3742). This virus-containing supernatant was used to infectNIH 3T3 cells, 10 times with 12 hour intervals. Control cells wereinfected in parallel with the insert-free vector virus obtained by thesame procedure. G418 selection showed that 100% of NIH 3T3 cells becameinfected with the virus. DNA from the infected cells was analyzyed bySouthern blot hybridization with a virus-specific probe. This analysisshowed that the infected cells contained multiple copies of theintegrated provirus.

Freshly-obtained multiply-infected NIH 3T3 cells were characterized by adecreased growth rate and plating efficiency. After several passages,however, their growth parameters became indistinguishable from thecontrol cells, suggesting the elimination of slowly growing cells fromthe population. At this stage, the cells were frozen and used for theexperiments described below.

EXAMPLE 8 Drug Resistance Pattern of NIH 3T3 Cells Carrying MultipleCopies of Anti-khcs GSEs

The infected cell population described in Example 7 were analyzed forresistance to several anticancer drugs by a growth inhibition assay. Forthis assay, 10⁴ cells per well were plated in 12-well plated and exposedto increasing concentrations of different drugs for 4 days. Relativecell numbers were measured by the methylene blue staining assay (Perryet al., 1992, Mutation Res. 286: 189-197). Despite the relativeinsensitivity of this assay when carried out with unselectedheterogeneous cell populations, infection with the virus carrying ananti-khcs GSE induced a pronounced increase in the resistance to thecytostatic effects of etoposide and amsacrine and, to a lesser extent,of Adriamycin camptothecin and cisplatin FIGS. 11A through 11C, FIG. 11Eand FIG. 11G. All of these drugs are known to induce DNA damage, albeitby different mechanism. Under the same assay conditions, no increase inresistance was observed with colchicine or actinomycin FIGS. 11D and11F.

To further characterize the nature of drug resistance conferred byanti-khcs, the above-described short-term growth inhibition assays werefollowed by long-term growth inhibition assays which measured both thecytostatic and the cytotoxic effects of different drugs. These assayswere carried out by incubating the cells for four days in the presenceof the drugs, followed by either two or six days in the absence of thedrug, to allow for programmed cell death, which is frequently associatedwith recovery from drug-induced inhibition (Kung et al., 1990, CancerRes. 50: 7307-7317). These assays showed that the GSE-carrying cellswere resistant to etoposide and adriamycin, but not to cisplatin,camptothecin or actinomycin D. Furthermore, the GSE-carrying cells werefound to be hypersensitive to colchicine and vinblastine, saidhypersensitivity being increasingly more evident with increasing lengthof the assay.

Thus, in the experiment shown in FIGS. 12A and 12B, NIH 3T3 cellscarrying the anti-khcs GSE and control cells without the GSE were platedat a density of 2×10⁴ per culture dish and grown for five days inconcentrations of either colchicine or vinblastine. The cells were thenfixed and stained, and the number of surviving cells determined andshown as a percentage of cell growth in the absence of drug. Theseresults clearly show that expression of the anti-khcs GSE in these cellswas accompanied by hypersensitivity to both colchicine and vinblastine.

To further investigate the discrepancy between the results obtained inthe short-term and long-term drug assays, analyses of the dynamics ofcell growth during and after treatment with 250 ng/mL etoposide, 20ng/mL camptothecin and 40 ng/mL colchicine were performed. In theseexperiments (shown in FIGS. 13A through 13D), NIH 3T3 cells were treatedwith the corresponding drugs for 5 days, and then incubated foradditional 6 days either in the presence or in the absence of the drug.The selected drug concentrations resulted in growth inhibition butlittle detectable cell death in continuous presence of the drug. After11 days of drug exposure the total cell number in the etoposide- orcamptothecin-treated populations of control cells was a little lowerthan after 5 days of drug exposure, but in the colchicine-treatedcontrol cell population a slow cell growth was still detectable. In theexperiments where the drug was removed after 5 days, cell growth wasinitially activated for all three drugs. After about two days of growthin the absence of the drug, the number of cells treated with etoposideor camptothecin decreased, however, due to extensive cell death, so thatthe final cell number in cell populations incubated in the absence ofthe drugs was practically the same as in the cells continuouslymaintained in the presence of colchicine or camptothecin. In contrast,cells pre-treated with colchicine undergo only limited cell death afterremoval from the drug, resulting in a relatively minor slowdown in cellgrowth tow days after removal from the drug, resulting major increase inthe total cell number in the populations removed from the drug relativeto those that were constantly maintained in colchicine FIGS. 13A through13D.

The expression of the anti-khcs GSE had different effects on the growthinhibition and recovery-associated cell death induced by these drugs. Incells treated with etoposide, anti-khcs decreased the growth-inhibitoryeffect of the drug to the same, relatively small extent after 5 or 11days of continuous exposure. In addition, the anti-khcs GSE decreasedthe amount of cell death in the populations released from etoposideinhibition. As a result, the increase in etoposide resistance conferredby this GSE was much more pronounced after release from the drug thanunder the conditions of continuous exposure (FIG. 13B). In cells treatedwith camptothecin, the GSE resulted in a small decrease of thegrowth-inhibitory effect of the drug, which was more pronounced after 5days of exposure but was reduced to negligible levels after 11 days ofcontinuous exposure FIG. 13C. The decrease in growth inhibition after 5days of exposure was accompanied by a slight increase in cell deathafter the removal from the drug, so that the GSE produced no significantlong-term difference in camptothecin resistance. In the populationstreated with colchicine, the anti-khcs GSE made cells more susceptibleto the growth-inhibitory effect of the drug; this effect was increasedwith prolonged exposure and was equally apparent in the populationsreleased from the drug after 5 days or continuously maintained incolchicine FIG. 13D.

The results of the above experiments indicated that the anti-khcs GSEacts by inhibiting the cytostatic effects of different DNA-damagingdrugs. In addition, this GSE appears to decrease the extent ofprogrammed cell death occurring after the release from the drug in cellstreated with some (etoposide) but not other (camptothecin) DNA-damagingagents.

The finding that cells carrying the anti-khcs GSE become hypersensitiveto the cytostatic effects of colchicine has potentially significanttherapeutic implications. The observed hypersensitivity to colchicine,an anti-microtubular agent, in cells with GSE-mediated inhibition ofkinesin is likely to be mechanically related to the essential functionsof kinesin, which moves various structures along the microtubules andmay be involved in the sliding of microtubules relative to each other,as well as the assembly and disassembly of microtubules. The pronouncedeffect of the anti-khcs GSE on the sensitivity of cells toanti-microtubule agents also indicates that this GSE affects the generalkinesin function in the cells, and is not limited to a particulardrug-response-specific isoform of kinesin. Since we have demonstratedthat down-regulation of the KHCS gene represents a natural mechanism ofdrug resistance in human tumor cells (see Example 11), thehypersensitivity to colchicine provides an approach to overcoming thistype of resistance in human cancer.

EXAMPLE 9 Assessment of Cellular Effects of Anti-kinesin GSEs

The virus carrying the anti-khcs GSE was treated for the ability toincrease the life span of primary mouse embryo fibroblasts (MEF). MEFwere prepared from 10 day old mouse embryos by a standard trypsinizationprocedure and senescent cells were frozen at different passages prior tocrisis. Senescent MEF, two weeks before crisis, were infected withrecombinant retroviruses carrying LNCX vector either without an insertor with anti-khcs. FIG. 14 shows MEF cell colonies two weeks aftercrisis. Relative to uninfected MEF cells, or cells infected with acontrol LNCX virus, cells infected with the anti-khcs showed a greatincrease in the proportion of cells surviving the crisis. Post-crisiscells infected with the anti-khcs virus showed no microscopicallyvisible features of neoplastic transformation. These results indicatethat anti-khcs promotes the immortalization of normal senescentfibroblasts. These results suggest that the normal function of kinesin-Smay be associated with the induction of programmed cell death occurringafter exposure to certain cytotoxic drugs or in the course of cellularsenescence. These results also indicate that isolation of GSEs thatconfer resistance to chemotherapeutic drugs can provide insight into thecellular genes and processes involved in cell growth regulation.

EXAMPLE 10 Biological Effects of Mouse Anti-khcs GSE on Human SenescentFibroblasts

The ability of the anti-khcs GSE described in Example 4 to promoteimmortalization of primary mouse embryo fibroblasts (demonstrated inExample 9) suggested that kinesin-S may act as a tumor suppressor bypreventing immortalization of normal mouse fibroblasts. To determine ifthis gene may play the same role in human cells, the ability of theanti-khcs GSE to affect the life span of primary human fibroblasts wasinvestigated.

Primary human fibroblasts, derived from human skin, were obtained fromthe Aging Cell Repository of the National Institutes of Aging. The cellswere grown in DMEM with 20% fetal calf serum supplemented with twice theconcentration of amino acids and vitamins normally used to supplementnormal human skin fibroblast growth in culture. Cells at the fifthpassage were infected with either the control pLNCX virus or the viruscarrying mouse anti-khcs GSE (produced as described above in Example 3).Four passages later, the control cells went into crisis, butGSE-carrying cells continued to grow (FIG. 15) and have so far survivedat least five additional passages. These results demonstrated that theanti-khcs GSE of this invention is also capable of prolonging life spanof primary human fibroblasts, indicating that KHCS is a potential tumorsuppressor in human cells.

EXAMPLE 11 Assessment of the Role of Decreased khcs Gene Expression inNaturally Occurring Mechanisms of Drug Resistance

To test whether decreased khcs gene expression is associated with anynaturally occurring mechanism of drug resistance, an assay was developedfor measuring khcs mRNA levels by cDNA-PCR. This assay is a modificationof the quantitative assay described by Noonan et al. (1990, Proc. Natl.Acad. Sci. USA 87: 7160-7164) for determining mdr-1 gene expression. Theoligonucleotide primers had the sequences AGTGGCTGGAAAACGAGCTA(SEQ.ID.NO.:5) and CTTGATCCCTTCTGGTTGAT (SEQ.ID.NO.:6). These primerswere used to amplify a 327 bp segment of mouse khcs cDNA, correspondingto the anti-khcs GSE. These primers efficiently amplified the mouse cDNAtemplate but not the genomic DNA, indicating that they spanned at leastone intron in the genomic DNA. Using these primers, we determined thatkhcs mRNA is expressed at a higher level in the mouse muscle tissue thanin the kidney, liver or spleen.

In another experiment a pair of primers amplifying a homologous segmentof the human KHCS cDNA was selected, based on the report human KHCsequence published by Navone et al. (1992, J. Cell. Biol. 117:1263-1275). The sequences of these primers are AGTGGCTTGAAAATGAGCTC(SEQ.ID.NO.:7) and CTTGATCCCTTCTGGTAGATG (SEQ.ID.NO.:8), and theyamplify a 327 bp cDNA fragment. These primers were used to test forchanges in the KHCS gene expression in several independently isolatedpopulations of human HeLa cells, each selected for spontaneouslyacquired etoposide resistance, β₂-microglobulin cDNA sequences wereamplified as an internal control. FIG. 16 shows the results of thecDNA-PCR assay on the following populations: CX(0), HeLa populationinfected with the LNCX vector virus and selected with G418; CX (200),the same cells selected for resistance to 200 ng/ml etoposide; Σ11(O),6(O), and Σ21(O), populations obtained after infection of HeLa cellswith recombinant retroviruses carrying different GSEs derived fromtopoisomerase α cDNA, as described in Example 1 of co-pending U.S.patent application Ser. No. 08/033,086, incorporated by reference, andselected with G418:Σ11 (1000), 6(1000) and Σ21(1000), the samepopulations selected for resistance to 1 μg/ml etoposide. As shown inFIG. 16, the yield of the PCR product specific for the khcs gene wassignificantly lower in each of the etoposide-selected populations thanin the control cells. This result indicates that a decrease in the khcsgene expression is a common natural mechanism for drug resistance.

EXAMPLE 12 Diagnostic Assay

The results presented in the above Examples suggest the utility ofdiagnostic assays for determining the expression levels of kinesin genesin tumor cells of a cancer patient, relative to a standardized set ofcell lines in vitro having well-characterized levels of kinesin heavychain gene expression correlated with their level of resistance tocertain chemotherapeutic drugs such as etoposide. One such standardizedset of cell lines comprise the HeLa cell lines described in Example 11.Alternatively, different, tissue-specific standardized set of cell linesare developed by drug selection for each cell type to be evaluated, forexample, using human K562 cells for evaluating patients having chronicmyelogenous leukemia, or human HL60 cells for patients having acutepromyelocytic leukemia.

The assay for kinesin would assess the appropriateness of treatment ofhuman cancer patients with certain anticancer therapeutic regimens.Patients whose tumor cells under-express kinesin may be refractory totreatment with DNA damaging agents, including radiation and thechemotherapeutic drugs etoposide, camptothecin, cisplatin andadriamycin. Such patients, however, may be particularly responsive totreatment with anti-microtubular agents such as colchicine, colcemide,vinblastine, vincristine or vindesine. On the other hand, patients whosetumor cells over-express kinesin, for example, may be responsive totreatment with DNA damaging agents and refractory to treatment withanti-microtubular agents. These assays provide, for the first time, abasis for making such therapeutic judgments before the fact, rather thanafter a therapeutic regimen has been tried and failed. The assay alsoprovide a basis for determining which patients, previously refractory totreatment with DNA damaging agents, particularly certain anticancerdrugs, would benefit from further chemotherapy using anti-microtubularagents, by distinguishing kinesin gene-mediated drug resistance fromother mechanism of drug resistance expected to result incross-resistance to both DNA damaging agents and anti-microtubulardrugs.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

8 20 base pairs nucleic acid single linear cDNA NO NO 1 AATCATCGATGGATGGATGG 20 23 base pairs nucleic acid single linear cDNA NO YES 2CCATCCATCC ATCGATGATT AAA 23 327 base pairs nucleic acid single linearcDNA NO YES 3 CTTGATCCCT TCTGGTTGAT GCCAGAAGCT CTTCCTGATC CAGCATTTGTATCTTCAATT 60 TCTCTACCAA TTGGCTTTGT TGGTTAATCT CTTCATCCTT GTCATCAAGTTGTTTATACA 120 ATTTAGCAAG TTCTTCTTCA CACTTTCTTC TTTCAGCATC GGTAAAACTACCAGCCATTC 180 CGACTGCAGC AGCTGGTTTA TCACTGGTAA TAGCAATATC TTTATCCGCTGTGAAGGCTT 240 CCAAATTAGC TTTCTCTTTG TCAAACTGCT CATCAATAGG CACTGTCTCCCCGTTACGCC 300 AACGGTTTAG CTCGTTTTCC AGCCACT 327 2389 base pairs nucleicacid single linear cDNA NO 4 CGACAAACAT CATCTGGGAA GACCCACACG ATGGAGGGTAAACTTCATGA TCCAGAAGGC 60 ATGGGAATTA TTCCAAGAAT AGTGCAAGAT ATTTTTAATTATATTTACTC CATGGATGAA 120 AATTTGGAAT TTCATATTAA GGTTTCATAT TTTGAAATATATTTGGATAA GATAAGGGAC 180 TTGTTAGATG TTTCAAAGAC TAACCTTTCA GTCCATGAAGACAAAAACCG TGTTCCCTAT 240 GTAAAGGGGT GCACAGAACG TTTCGTGTGT AGTCCAGATGAAGTCATGGA TACCATAGAT 300 GAAGGGAAAT CCAACAGAGA TGTCGCAGTT ACAAATATGAATGAACATAG CTCTAGGAGC 360 CACAGCATAT TTCTTATTAA TGTAAAACAA GAGAATACACAAACGGAACA GAAACTCAGT 420 GGAAAGCTTT ATCTGGTTGA TTTAGCTGGC AGTGAGAAGGTTAGTAAGAC TGGGGCTGAA 480 GGTGCTGTGC TGGATGAAGC TAAGAACATC AAGAAGTCACTTTCTGCACT TGGAAATGTC 540 ATTTCTGCTT TGGCAGAGGG CAGTACCTAT GTTCCTTATCGAGATAGTAA AATGACCAGA 600 ATTCTTCAAG ATTCATTAGG TGGCAACTGT AGGACCACTATTGTCATATG CTGCTCTCCA 660 TCATCATACA ATGAGTCTGA GACAAAGTCA ACACTCCTCTTTGGTCAAAG GGCCAAAACA 720 ATTAAGAACA CAGTCTGTGT CAATGTAGAG TTAACTGCAGAGCAGTGGAA AAAGAAGTAT 780 GAAAAAGAAA AGGAAAAAAA TAAGACTCTA CGGAACACTATTCAGTGGCT GGAAAACGAG 840 CTAAACCGTT GGCGTAACGG GGAGACAGTG CCTATTGATGAGCAGTTTGA CAAAGAGAAA 900 GCTAATTTGG AAGCCTTCAC AGCGGATAAA GATACTGCTATTACCAGTGA TAAACCAGCT 960 GCTGCAGTCG GAATGGCTGG TAGTTTTACC GATGCTGAAAGAAGAAAGTG TGAAGAAGAA 1020 CTTGCTAAAT TGTATAAACA GCTTGATGAC AAGGATGAAGAGATTAACCA ACAAAGCCAA 1080 TTGGTAGAGA AATTGAAGAC ACAAATGCTG GATCAGGAAGAGCTTCTGGC ATCAACCAGA 1140 AGGGATCAAG ATAATATGCA AGCTGAACTG AATCGCCTCCAAGCAGAAAA TGATGCTTCT 1200 AAAGAAGAAG TCAAAGAAGT TTTACAGGCC TTAGAGGAACTGGCTGTTAA TTATGATCAG 1260 AAGTCTCAGG AAGTTGAAGA CAAAACAAAG GAATATGAATTGCTTAGTGA TGAATTGAAT 1320 CAAAAATCTG CAACTTTAGC AAGTATTGAT GCTGAGCTTCAGAAGCTGAA GGAAATGACC 1380 AACCACCAGA AGAAACGAGC AGCTGAAATG ATGGCATCATTATTAAAAGA CCTTGCAGAA 1440 ATAGGAATTG CTGTGGGGAA TAACGATGTG AAGCAACCAGAAGGAACTGG TATGATAGAT 1500 GAAGAGTTTA CTGTTGCAAG ACTCTACATT AGCAAAATGAAATCAGAAGT AAAGACCATG 1560 GTGAAACGCT GCAAACAGCT AGAAAGCACG CAGACTGAGAGCAACAAAAA AATGGAAGAA 1620 AATGAGAAAG AGTTAGCAGC ATGCCAGCTT CGGATCTCCCAACATGAAGC CAAAATCAAG 1680 TCACTGACTG AGTACCTTCA GAATGTAGAA CAAAAGAAGAGGCAGCTGGA GGAATCTGTT 1740 GATTCCCTTG GTGAGGAGCT AGTCCAACTC CGAGCACAAGAGAAAGTCCA TGAAATGGAA 1800 AAAGAGCACT TGAACAAGGT TCAGACTGCA AATGAAGTCAAGCAAGCTGT TGAGCAGCAG 1860 ATCCAGAGTC ACAGAGAAAC CCACCAAAAA CAAATCAGTAGCTTGCGAGA TGAAGTTGAG 1920 GCAAAGGAAA AGCTAATCAC TGACCTCCAA GACCAAAACCAGAAGATGGT GTTGGAGCAG 1980 GAACGGCTAA GGGTGGAGCA TGAGAGGCTG AAGGCTACAGACCAAGAGAA GAGCAGGAAG 2040 CTGCATGAGC TCACGGTTAT GCAAGACAGA CGAGAACAAGCAAGACAAGA CTTGAAGGGT 2100 TTGGAGGAGA CCGTGGCAAA AGAACTTCAG ACTTTACACAACCTGCGTAA GCTCTTTGTT 2160 CAGGACTTGG CTACCAGGGT GAAAAAGAGG CCGAGGTCGACTCTGACGAC ACTGGCGGCA 2220 GTGCTGCACA GAAGCAGAAA ATCTCCTTCC TTGAAAACAACCTTGAACAG CTCACCAAAG 2280 TGCACAAGCA GTTGGTACGT GATAATGCAG ATCTTCGCTGTGAGCTTCCT AAGTTAGAGA 2340 AACGGCTTAG AGCTACTGCA GAAAGAGTGA AAGCTTTGGAGTCAGCCCG 2389 22 base pairs nucleic acid single linear cDNA NO NO 5CTCCCAAGCT TATGGATGGA TG 22 25 base pairs nucleic acid single linearcDNA NO YES 6 CATCCATCCA TAAGCTTGGG AGAAA 25 24 base pairs nucleic acidsingle linear cDNA NO 7 TGAGTGAGTG AATCGATGAT TAAA 24 21 base pairsnucleic acid single linear cDNA NO YES 8 AATCATCGAT TCACTCACTC A 21

What is claimed is:
 1. A diagnostic assay for determining whethermalignant cells in a tumor or a tissue in an animal are sensitive orresistant to DNA damaging chemotherapeutic drugs or agents, the methodcomprising the steps of (a) isolating cellular RNA comprising messengerRNA from malignant cells of a tumor or a tissue from an animal; (b)measuring a level of expression of a kinesin gene in the malignant cellsof the tumor or tissue; (c) determining whether the level of expressionof the kinesin gene in the malignant cells of the tumor or tissueindicates that the kinesin gene is over-expressed or under-expressed inthe malignant cells of the tumor or tissue in the animal compared withexpression of said kinesin gene in non-malignant cells or tissue; and(d) providing a diagnosis that malignant cells in said tumor or tissueare resistant to DNA damaging chemotherapeutic drugs or agents when thekinesin gene is under expressed compared with non-malignant cells ortissue or providing a diagnosis that malignant cells in said tumor ortissue are sensitive to DNA damaging chemotherapeutic drugs or agentswhen the kinesin gene is over-expressed in the tumor or tissue comparedwith non-malignant cells or tissue.
 2. A diagnostic assay fordetermining whether malignant cells in a tumor or a tissue in an animalare sensitive or resistant to DNA damaging chemotherapeutic drugs oragents, the method comprising the steps of (a) isolating cellularprotein from malignant cells of a tumor or a tissue from an animal; (b)measuring an amount of a kinesin protein in the malignant cells of thetumor or tissue of the animal; (c) determining whether the amount of thekinesin protein in the malignant cells of the tumor or tissue indicatesthat the kinesin gene is over-expressed or under-expressed in themalignant cells of the tumor or tissue in the animal compared with theamount of said kinesin protein in non-malignant cells or tissue; and (d)providing a diagnosis that malignant cells in said tumor or tissue areresistant to DNA damaging chemotherapeutic drugs or agents when theamount of kinesin protein is decreased in the tumor or malignant cellscompared with non-malignant cells or tissue, or providing a diagnosisthat malignant cells in the tumor or tissue are sensitive to DNAdamaging chemotherapeutic drugs or agents when the amount of kinesinprotein is increased in the malignant cells of the tumor or tissuecompared with non-malignant cells or tissues.
 3. A diagnostic assayaccording to claim 1 or 2, wherein the diagnosis is that the malignantcells of the tissue or tumor in the animal will be resistant to ananticancer drug that is a DNA damaging agent where the kinesin gene isunder-expressed in the malignant cells of the tumor.
 4. A diagnosticassay according to claim 1 or 2, wherein the diagnosis is that themalignant cells of the tissue or tumor in the animal will be susceptibleto an anticancer drug that is a DNA damaging agent where the kinesingene is over-expressed in the malignant cells or tumor.
 5. A diagnosticassay for determining whether malignant cells in a tumor or a tissue inan animal are sensitive or resistant to antimicrotubule-directedchemotherapeutic drugs or agents, the method comprising the steps of (a)isolating cellular RNA comprising messenger RNA from malignant cells ofa tumor or a tissue from an animal; (b) measuring a level of expressionof a kinesin gene in the malignant cells of the tumor or tissue; (c)determining whether the level of expression of the kinesin gene in themalignant cells of the tumor or tissue indicates that the kinesin geneis over-expressed or under-expressed in the malignant cells of the tumoror tissue in the animal compared with expression of said kinesin gene innon-malignant cells or tissue; and (d) providing a diagnosis thatmalignant cells in said tumor or tissue are sensitive toantimicrotubule-directed chemotherapeutic drugs or agents when thekinesin gene is under expressed compared with non-malignant cells ortissue or providing a diagnosis that malignant cells in said tumor ortissue are resistant to antimicrotubule-directed chemotherapeutic drugsor agents when the kinesin gene is over-expressed in the tumor or tissuecompared with non-malignant cells or tissues.
 6. A diagnostic assay fordetermining whether malignant cells in a tumor or a tissue in an animalare sensitive or resistant to antimicrotubule-directed chemotherapeuticdrugs or agents, the method comprising the steps of (a) isolatingcellular protein from malignant cells of a tumor or a tissue from ananimal; (b) measuring an amount of a kinesin protein in the malignantcells of the tumor or tissue of the animal; (c) determining whether theamount of kinesin protein in the malignant cells of the tumor or tissueindicates that the kinesin gene is over-expressed or under-expressed inthe malignant cells of the tumor or tissue in the animal compared withthe amount of said kinesin protein in non-malignant cells or tissue; and(d) providing a diagnosis that malignant cells in said tumor or tissueare sensitive to antimicrotubule-directed chemotherapeutic drugs oragents when the amount of kinesin protein is decreased in the tumor ormalignant cells compared with non-malignant cells or tissue, orproviding a diagnosis that malignant cells in the tumor or tissue areresistant to antimicrotubule-directed chemotherapeutic drugs or agentswhen the amount of kinesin protein is increased in the malignant cellsof the tumor or tissue compared with non-malignant cells or tissues. 7.A diagnostic assay according to claim 5 or 6, wherein the diagnosis isthat the malignant cells of the tissue or tumor in the animal will beresistant to an anticancer drug that is an anti-microtubule agent wherethe kinesin gene is over-expressed in the malignant cells or tumor.
 8. Adiagnostic assay according to claim 5 or 6, wherein the diagnosis isthat the malignant cells of the tissue or tumor in the animal will besusceptible to an anticancer drug that is an anti-microtubule agentwhere the kinesin gene is under-expressed in the malignant cells ortumor.